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Abstract Background The discovery of viruses in small mammalian populations, particularly rodents, has expanded the family Paramyxoviridae. The overlap in habitats between rodents and humans increases the risk of zoonotic events, underscoring the importance of active surveillance. Rodent species, such as Apodemus agrarius, are natural hosts for Paramyxoviridae in the Republic of Korea (ROK). However, it is unknown whether Paramyxoviridae is present in Micromys minutus, another common rodent. Method Here, we screened M. minutus collected from the Gangwon Province in the ROK for paramyxoviruses using nested polymerase chain reaction and confirm positive samples by next-generation metagenomic sequencing. Complete paramyxovirus genomes were further characterized by phylogenetic analysis, amino acid similarity, secondary structure, and cophylogeny. Result Overall, 57 of 145 (39.3%) M. minutus kidney samples tested positive for paramyxoviruses. Among them, four whole genome sequences were identified and clustered within the genus Jeilongvirus. One sequence was determined as Samak Micromys paramyxovirus 1 (SMPV-1; 19,911 nucleotides long) and three sequences as Samak Micromys paramyxovirus 2 (SMPV-2; 18,199 nucleotides long). SMPV-1 has a smaller hydrophobic gene and a longer glycoprotein gene than SMPV-2. Cophylogenetic analysis suggests that SMPV-1 evolved through co-divergence, whereas SMPV-2 was inferred to have undergone transfer events. Conclusion These findings highlight the prevalence of paramyxoviruses in the wild and the potential of M. minutus as a natural viral reservoir. The discovery of SMPV-1 and SMPV − 2 also reveals the genetic diversity and evolutionary history of the genus Jeilongvirus in the Paramyxoviridae.

Seoul (SEOV) and Hantaan (HTNV) orthohantaviruses are significant zoonotic pathogens responsible for hemorrhagic fever with renal syndrome. Here, we investigated the molecular evolution of SEOV and HTNV through phylogenetic and bioinformatic analyses using complete genome sequences of their large (L), medium (M), and small (S) gene segments. Despite similar epizootic cycles and clinical symptoms, SEOV and HTNV exhibited distinct genetic and evolutionary dynamics. The phylogenetic trees of each segment consistently showed major genetic clades associated with the geographical distribution of both viruses. Remarkably, SEOV M and S segments exhibit higher evolutionary rates, rapidly increasing genetic diversity, and a more recent origin in contrast to HTNV. Reassortment events were infrequent, but both viruses appear to utilize the M gene segment in genetic exchanges. SEOV favors the L or M segment reassortment, while HTNV prefers the M or S segment exchange. Purifying selection dominates in all three gene segments of both viruses, yet SEOV experiences an elevated positive selection in its glycoprotein Gc ectodomain. Key amino acid differences, including a positive ‘lysine fence’ (through residues K77, K82, K231, K307, and K310) located at the tip of the Gn, alongside the physical stability around an RGD-like motif through M108-F334 interaction, may contribute to the unique antigenic properties of SEOV. With the increasing global dispersion and potential implications of SEOV for the global public health landscape, this study highlights the unique evolutionary dynamics and antigenic properties of SEOV and HTNV in informing vaccine design and public health preparedness.

ABSTRACT: Objectives: To investigate the risk of haematologic and solid organ malignancies in patients with haemorrhagic fever with renal syndrome (HFRS) compared with the general population. Methods: This propensity-score-matched cohort study was conducted using data collected from the Korean national health insurance service (NHIS) between January 2003 and December 2017. The HFRS cohort included 5888 newly diagnosed cases of HFRS, and 412,804 general participants from the NHIS database were included as the control cohort. The incidence rate of malignancies was assessed and compared between the HFRS and control cohorts. Results: There were 64 cases of haematologic malignancy in 236,286 person-years of observation, and 1245 cases of solid organ cancer in 209,333 person-years. The risks of haematologic malignancy and solid organ cancer were significantly higher in the HFRS cohort [adjusted hazards ratio (aHR) 4.10, 95% confidence interval (CI) 2.36–7.14] than the control cohort [aHR 2.97, 95% CI 2.60–3.38). In subgroup analysis, the HFRS cohort was associated with high hazard ratios for leukaemia and non-Hodgkin lymphoma. The HFRS cohort also had increased aHRs for all types of solid organ cancer. Conclusions: Patients with HFRS are at increased risk of both haematologic and solid organ malignancies compared with the general population, and this increased proportionally over time. Careful monitoring for malignancy after the onset of HFRS may be necessary.

BackgroundWhole-genome sequencing plays a critical role in the genomic epidemiology intended to improve understanding the spread of emerging viruses. Dabie bandavirus, causing severe fever with thrombocytopenia syndrome (SFTS), is a zoonotic tick-borne virus that poses a significant public health threat. We aimed to evaluate a novel amplicon-based nanopore sequencing tool to obtain whole-genome sequences of Dabie bandavirus, also known as SFTS virus (SFTSV), and investigate the molecular prevalence in wild ticks, Republic of Korea (ROK).Principal findingsA total of 6,593 ticks were collected from Gyeonggi and Gangwon Provinces, ROK in 2019 and 2020. Quantitative polymerase chain reaction revealed the presence of SFSTV RNA in three Haemaphysalis longicornis ticks. Two SFTSV strains were isolated from H. longicornis captured from Pocheon and Cheorwon. Multiplex polymerase chain reaction-based nanopore sequencing provided nearly full-length tripartite genome sequences of SFTSV within one hour running. Phylogenetic and reassortment analyses were performed to infer evolutionary relationships among SFTSVs. Phylogenetic analysis grouped SFTSV Hl19-31-4 and Hl19-31-13 from Pocheon with sub-genotype B-1 in all segments. SFTSV Hl20-8 was found to be a genomic organization compatible with B-1 (for L segment) and B-2 (for M and S segments) sub-genotypes, indicating a natural reassortment between sub-genotypes.Conclusion/significanceAmplicon-based next-generation sequencing is a robust tool for whole-genome sequencing of SFTSV using the nanopore platform. The molecular prevalence and geographical distribution of SFTSV enhanced the phylogeographic map at high resolution for sophisticated prevention of emerging SFTS in endemic areas. Our findings provide important insights into the rapid whole-genome sequencing and genetic diversity for the genome-based diagnosis of SFTSV in the endemic outbreak.

Whole-genome sequencing provides a robust platform for investigating the epidemiology and transmission of emerging viruses. Oxford Nanopore Technologies allows for real-time viral sequencing on a local laptop system for point-of-care testing. Seoul orthohantavirus (Seoul virus, SEOV), harbored by Rattus norvegicus and R. rattus, causes mild hemorrhagic fever with renal syndrome and poses an important threat to public health worldwide. We evaluated the deployable MinION system to obtain high-fidelity entire-length sequences of SEOV for the genome identification of accurate infectious sources and their genetic diversity. One-step amplicon-based nanopore sequencing was performed from SEOV 80–39 specimens with different viral copy numbers and SEOV-positive wild rats. The KU-ONT-SEOV-consensus module was developed to analyze SEOV genomic sequences generated from the nanopore system. Using amplicon-based nanopore sequencing and the KU-ONT-consensus pipeline, we demonstrated novel molecular diagnostics for acquiring full-length SEOV genome sequences, with sufficient read depth in less than 6 h. The consensus sequence accuracy of the SEOV small, medium, and large genomes showed 99.75–100% (for SEOV 80–39 isolate) and 99.62–99.89% (for SEOV-positive rats) identities. This study provides useful insights into on-site diagnostics based on nanopore technology and the genome epidemiology of orthohantaviruses for a quicker response to hantaviral outbreaks.

BackgroundHantavirus infection occurs through the inhalation of aerosolized excreta, including urine, feces, and saliva of infected rodents. The presence of Hantaan virus (HTNV) RNA or infectious particles in urine specimens of patient with hemorrhagic fever with renal syndrome (HFRS) remains to be investigated.Methodology/principal findingsWe collected four urine and serum specimens of Republic of Korea Army (ROKA) patients with HFRS. We performed multiplex PCR-based next-generation sequencing (NGS) to obtain the genome sequences of clinical HTNV in urine specimens containing ultra-low amounts of viral genomes. The epidemiological and phylogenetic analyses of HTNV demonstrated geographically homogenous clustering with those in Apodemus agrarius captured in highly endemic areas, indicating that phylogeographic tracing of HTNV genomes reveals the potential infection sites of patients with HFRS. Genetic exchange analyses showed a genetic configuration compatible with HTNV L segment exchange in nature.Conclusion/significanceOur results suggest that whole or partial genome sequences of HTNV from the urine enabled to track the putative infection sites of patients with HFRS by phylogeographically linking to the zoonotic HTNV from the reservoir host captured at endemic regions. This report raises awareness among physicians for the presence of HTNV in the urine of patients with HFRS.

Seoul virus (SEOV), an etiological agent for hemorrhagic fever with renal syndrome, poses a significant public health threat worldwide. This study evaluated the feasibility of a mobile Biomeme platform for facilitating rapid decision making of SEOV infection. A total of 27 Rattus norvegicus were collected from Seoul Metropolitan City and Gangwon Province in Republic of Korea (ROK), during 2016–2020. The serological and molecular prevalence of SEOV was 5/27 (18.5%) and 2/27 (7.4%), respectively. SEOV RNA was detected in multiple tissues of rodents using the Biomeme device, with differences in Ct values ranging from 0.6 to 2.1 cycles compared to a laboratory benchtop system. Using amplicon-based next-generation sequencing, whole-genome sequences of SEOV were acquired from lung tissues of Rn18-1 and Rn19-5 collected in Gangwon Province. Phylogenetic analysis showed a phylogeographical diversity of rat-borne orthohantavirus collected in Gangwon Province. We report a novel isolate of SEOV Rn19-5 from Gangwon Province. Our findings demonstrated that the Biomeme system can be applied for the molecular diagnosis of SEOV comparably to the laboratory-based platform. Whole-genome sequencing of SEOV revealed the phylogeographical diversity of orthohantavirus in the ROK. This study provides important insights into the field-deployable diagnostic assays and genetic diversity of orthohantaviruses for the rapid response to hantaviral outbreaks in the ROK.

BackgroundOrthohantaviruses, causing hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome, pose a significant public health threat worldwide. Despite the significant mortality and morbidity, effective antiviral therapeutics for orthohantavirus infections are currently unavailable. This study aimed to investigate the prevalence of HFRS-associated orthohantaviruses and identify the etiological agent of orthohantavirus outbreaks in southern Republic of Korea (ROK).Methodology/principal findingsWe collected small mammals on Jeju Island during 2018-2020. We detected the Hantaan virus (HTNV)-specific antibodies and RNA using an indirect immunofluorescence assay test and reverse transcription-polymerase chain reaction on Apodemus agrarius chejuensis (A. chejuensis). The prevalence of anti-HTNV antibodies among rodents was 14.1%. A total of six seropositive mouse harbored HTNV RNA. The amplicon-based next-generation sequencing provided nearly full-length tripartite genomic sequences of six HTNV harbored by A. chejuensis. Phylogenetic and tanglegram analyses were conducted for inferring evolutionary relationships between orthohantaviruses with their reservoir hosts. Phylogenetic analysis showed a novel distinct HTNV genotype. The detected HTNV genomic sequences were phylogenetically related to a viral sequence derived from HFRS patient in southern ROK. Tanglegram analysis demonstrated the segregation of HTNV genotypes corresponding to Apodemus spp. divergence.Conclusions/significanceOur results suggest that A. chejuensis-borne HTNV may be a potential etiological agent of HFRS in southern ROK. Ancestral HTNV may infect A. chejuensis prior to geological isolation between the Korean peninsula and Jeju Island, supporting the co-evolution of orthohantaviruses and rodents. This study arises awareness among physicians for HFRS outbreaks in southern ROK.

Of various rodent-borne hantaviruses, Seoul orthohantavirus (SEOV) causes haemorrhagic fever with renal syndrome (HFRS), as does Hantaan orthohantavirus (HTNV). Given global-scale of cases of human infection with SEOV, it is of great clinical importance to distinguish SEOV from other HFRS-causing hantaviruses. In May 2019, a middle-aged patient who had lived in a suburban area of Chungcheong Province, Republic of Korea and enjoyed outdoor activities was transferred to Asan Medical Center in Seoul, Republic of Korea with HFRS; his symptoms included high fever and generalized myalgia. The rapid diagnostic test performed immediately after his transfer detected HTNV-specific antibodies, and the patient was treated accordingly. However, two consecutive IFAs performed at ten-day intervals showed no HTNV-specific immunoglobulin (Ig) G. During continuous supportive care, next-generation sequencing successfully identified viral genomic sequences in the patient's serum, which were SEOV and not HTNV. Phylogenetic analysis grouped the L, M, and S genes of this SEOV strain together with those of rat- or human-isolated Korean strains reported previously. Given global outbreaks and public health threats of zoonotic hantaviruses, a causative pathogen of hantavirus HFRS should be identified correctly at the time of diagnosis and by point-of-care testing.

Emerging and re-emerging RNA viruses pose significant public health, economic, and societal burdens. Hantaviruses (genus Orthohantavirus, family Hantaviridae, order Bunyavirales) are enveloped, negative-sense, single-stranded, tripartite RNA viruses that are emerging zoonotic pathogens harbored by small mammals such as rodents, bats, moles, and shrews. Orthohantavirus infections cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome in humans (HCPS). Active targeted surveillance has elucidated high-resolution phylogeographic relationships between patient- and rodent-derived orthohantavirus genome sequences and identified the infection source by temporally and spatially tracking viral genomes. Active surveillance of patients with HFRS entails 1) recovering whole-genome sequences of Hantaan virus (HTNV) using amplicon (multiplex PCR-based) next-generation sequencing, 2) tracing the putative infection site of a patient by administering an epidemiological questionnaire, and 3) collecting HTNV-positive rodents using targeted rodent trapping. Moreover, viral genome tracking has been recently performed to rapidly and precisely characterize an outbreak from the emerging virus. Here, we reviewed genomic epidemiological and active surveillance data for determining the emergence of zoonotic RNA viruses based on viral genomic sequences obtained from patients and natural reservoirs. This review highlights the recent studies on tracking viral genomes for identifying and characterizing emerging viral outbreaks worldwide. We believe that active surveillance is an effective method for identifying rodent-borne orthohantavirus infection sites, and this report provides insights into disease mitigation and preparedness for managing emerging viral outbreaks.

BackgroundHantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius (the striped field mouse), causes hemorrhagic fever with renal syndrome (HFRS) in humans. Viral genome-based surveillance at new expansion sites to identify HFRS risks plays a critical role in tracking the infection source of orthohantavirus outbreak. In the Republic of Korea (ROK), most studies demonstrated the serological prevalence and genetic diversity of orthohantaviruses collected from HFRS patients or rodents in Gyeonggi Province. Gangwon Province is a HFRS-endemic area with a high incidence of patients and prevalence of infected rodents, ROK. However, the continued epidemiology and surveillance of orthohantavirus remain to be investigated.Methodology/principal findingsWhole-genome sequencing of HTNV was accomplished in small mammals collected in Gangwon Province during 2015-2018 by multiplex PCR-based next-generation sequencing. To elucidate the geographic distribution and molecular diversity of viruses, we conducted phylogenetic analyses of HTNV tripartite genomes. We inferred the hybrid zone using cline analysis to estimate the geographic contact between two different HTNV lineages in the ROK. The graph incompatibility based reassortment finder performed reassortment analysis. A total of 12 HTNV genome sequences were completely obtained from A. agrarius newly collected in Gangwon Province. The phylogenetic and cline analyses demonstrated the genetic diversity and hybrid zone of HTNV in the ROK. Genetic exchange analysis suggested the possibility of reassortments in Cheorwon-gun, a highly HFRS-endemic area.Conclusions/significanceThe prevalence and distribution of HTNV in HFRS-endemic areas of Gangwon Province enhanced the phylogeographic map for orthohantavirus outbreak monitoring in ROK. This study revealed the hybrid zone reflecting the genetic diversity and evolutionary dynamics of HTNV circulating in Gangwon Province. The results arise awareness of rodent-borne orthohantavirus diseases for physicians in the endemic area of ROK.

The ability to accurately predict the early progression of hemorrhagic fever with renal syndrome (HFRS) is crucial for reducing morbidity and mortality rates in severely affected patients. However, the utility of biomarkers for predicting clinical outcomes remains elusive in HFRS. The aims of the current study were to analyze the serum levels of immune function-related proteins and identify novel biomarkers that may help ascertain clinical outcomes of HFRS. Enzyme-linked immunosorbent assay, Luminex, and bioanalyzer assays were used to quantitatively detect 15 biomarkers in 49 serum samples of 26 patients with HFRS. High hemoglobin (HGB) and low urine output (UO) levels were identified as potential biomarkers associated with the acute HFRS. The serum soluble urokinase plasminogen activator receptor (suPAR) and C-X-C motif chemokine ligand 10 (CXCL10) values increased in the early phase of diseases. Elevated suPAR, interleukin-10 (IL-10), CXCL10, and decreased transforming growth factor-beta 3 (TGF-β3) were representative predictors of the disease severity. Upregulation of the HGB showed a significant correlation with high levels of suPAR and CXCL10. Reduced UO positively correlated with increased suPAR, CXCL10, and TGF-β2, and decreased vascular endothelial growth factor and TGF-β3. The changing HGB and UO criteria, high suPAR, IL-10, CXCL10, and low TGF-β3 of HFRS raise significant awareness for physicians regarding prospective biomarkers for monitoring early warning signs of HFRS. This study provides critical insights into the clinical and immunological biomarkers for disease severity and progression in patients with HFRS to identify early predictions of fatal outcomes.

Hepatitis A virus (HAV) is a serious threat to public health worldwide. We used multiplex polymerase chain reaction (PCR)-based next-generation sequencing (NGS) to derive information on viral genetic diversity and conduct precise phylogenetic analysis. Four HAV genome sequences were obtained using multiplex PCR-based NGS. HAV whole-genome sequence of one sample was obtained by conventional Sanger sequencing. The HAV strains demonstrated a geographic cluster with sub-genotype IA strains in the Republic of Korea. The phylogenetic pattern of HAV viral protein (VP) 3 region showed no phylogenetic conflict between the whole-genome and partial-genome sequences. The VP3 region in serum and stool samples showed sensitive detection of HAV with differences of quantification that did not exceed

Seoul virus (SEOV) poses a worldwide public health threat. This virus, which is harbored by Rattus norvegicus and R. rattus rats, is the causative agent of hemorrhagic fever with renal syndrome (HFRS) in humans, which has been reported in Asia, Europe, the Americas, and Africa. Defining SEOV genome sequences plays a critical role in development of preventive and therapeutic strategies against the unique worldwide hantavirus. We applied multiplex PCR–based next-generation sequencing to obtain SEOV genome sequences from clinical and reservoir host specimens. Epidemiologic surveillance of R. norvegicus rats in South Korea during 2000–2016 demonstrated that the serologic prevalence of enzootic SEOV infections was not significant on the basis of sex, weight (age), and season. Viral loads of SEOV in rats showed wide dissemination in tissues and dynamic circulation among populations. Phylogenetic analyses showed the global diversity of SEOV and possible genomic configuration of genetic exchanges.

Paramyxoviruses, negative-sense single-stranded RNA viruses, pose a critical threat to human public health. Currently, 78 species, 17 genera, and 4 subfamilies of paramyxoviruses are harbored by multiple natural reservoirs, including rodents, bats, birds, reptiles, and fish. Henipaviruses are critical zoonotic pathogens that cause severe acute respiratory distress and neurological diseases in humans. Using reverse transcription-polymerase chain reaction, 115 Crocidura species individuals were examined for the prevalence of paramyxovirus infections. Paramyxovirus RNA was observed in 26 (22.6%) shrews collected at five trapping sites, Republic of Korea. Herein, we report two genetically distinct novel paramyxoviruses (genus: Henipavirus): Gamak virus (GAKV) and Daeryong virus (DARV) isolated from C. lasiura and C. shantungensis, respectively. Two GAKVs and one DARV were nearly completely sequenced using next-generation sequencing. GAKV and DARV contain six genes (3′-N-P-M-F-G-L-5′) with genome sizes of 18,460 nucleotides and 19,471 nucleotides, respectively. The phylogenetic inference demonstrated that GAKV and DARV form independent genetic lineages of Henipavirus in Crocidura species. GAKV-infected human lung epithelial cells elicited the induction of type I/III interferons, interferon-stimulated genes, and proinflammatory cytokines. In conclusion, this study contributes further understandings of the molecular prevalence, genetic characteristics and diversity, and zoonotic potential of novel paramyxoviruses in shrews.

Abstract It has been noticed that neuraminidase (NA) stalk truncation has arisen from evolutionary adaptation of avian influenza A viruses (IAVs) from wild aquatic birds to domestic poultry. We identified this molecular alteration after the adaptation of a 2009 pandemic H1N1 virus (pH1N1) in BALB/c mice. The mouse-adapted pH1N1 lost its eight consecutive amino acids including one potential N-linked glycosite from the NA stalk region. To explore the relationship of NA stalk truncation or deglycosylation with viral pathogenicity changes, we generated NA stalk mutant viruses on the pH1N1 backbone by reverse genetics. Intriguingly, either NA stalk truncation or deglycosylation changed pH1N1 into a lethal virus to mice by resulting in extensive pathologic transformation in the mouse lungs and systemic infection affecting beyond the respiratory organs in mice. The increased pathogenicity of these NA stalk mutants was also reproduced in ferrets. In further investigation using a human-infecting H7N9 avian IAV strain, NA stalk truncation or deglycosylation enhanced the replication property and pathogenicity of H7N9 NA stalk mutant viruses in the same mouse model. Taken together, our results suggest that NA stalk truncation or deglycosylation can be the pathogenic determinants of seasonal influenza viruses associated with the evolutionary adaptation of IAVs.

Whole-genome sequencing of infectious agents enables the identification and characterization of emerging viruses. The MinION device is a portable sequencer that allows real-time sequencing in fields or hospitals. Hantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius, causes hemorrhagic fever with renal syndrome (HFRS) and poses a critical public health threat worldwide. In this study, we aimed to evaluate the feasibility of using nanopore sequencing for whole-genome sequencing of HTNV from samples having different viral copy numbers. Amplicon-based next-generation sequencing was performed in A. agrarius lung tissues collected from the Republic of Korea. Genomic sequences of HTNV were analyzed based on the viral RNA copy numbers. Amplicon-based nanopore sequencing provided nearly full-length genomic sequences of HTNV and showed sufficient read depth for phylogenetic analysis after 8 h of sequencing. The average identity of the HTNV genome sequences for the nanopore sequencer compared to those of generated from Illumina MiSeq revealed 99.8% (L and M segments) and 99.7% (S segment) identities, respectively. This study highlights the potential of the portable nanopore sequencer for rapid generation of accurate genomic sequences of HTNV for quicker decision making in point-of-care testing of HFRS patients during a hantavirus outbreak.

An epidemiological investigation was conducted for a scrub typhus case reported in a U.S. Forces Korea military patient in the Republic of Korea in November 2018. The patient had a fever, maculopapular rash, and an eschar. The full-length sequence of Orientia tsutsugamushi 56-kDa type-specific antigen (TSA) gene was obtained from eschar tissue by multiplex PCR-based Next Generation Sequencing for genetic identification. Based on the 56-kDa TSA gene, the O. tsutsugamushi aligned most closely with the Boryong strain.

The virus behind the current pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the etiology of novel coronavirus disease (COVID-19) and poses a critical public health threat worldwide. Effective therapeutics and vaccines against multiple coronaviruses remain unavailable. Single-chain variable fragment (scFv), a recombinant antibody, exhibits broad-spectrum antiviral activity against DNA and RNA viruses owing to its nucleic acid-hydrolyzing property. The antiviral activity of 3D8 scFv against SARS-CoV-2 and other coronaviruses was evaluated in Vero E6 cell cultures. Viral growth was quantified with quantitative RT-qPCR and plaque assay. The nucleic acid-hydrolyzing activity of 3D8 was assessed through abzyme assays of in vitro viral transcripts and cell viability was determined by MTT assay. We found that 3D8 inhibited the replication of SARS-CoV-2, human coronavirus OC43 (HCoV-OC43), and porcine epidemic diarrhea virus (PEDV). Our results revealed the prophylactic and therapeutic effects of 3D8 scFv against SARS-CoV-2 in Vero E6 cells. Immunoblot and plaque assays showed the reduction of coronavirus nucleoproteins and infectious particles, respectively, in 3D8 scFv-treated cells. These data demonstrate the broad-spectrum antiviral activity of 3D8 against SARS-CoV-2 and other coronaviruses. Thus, it could be considered a potential antiviral countermeasure against SARS-CoV-2 and zoonotic coronaviruses.

Adenoviruses (family Adenoviridae) infect various organ systems and cause diseases in a wide range of host species. In this study, we examined multiple tissues from Chinstrap penguins (Pygoscelis antarctica), collected in Antarctica during 2009 and 2010, for the presence of novel adenoviruses by PCR. Analysis of a 855-bp region of the hexon gene of a newly identified adenovirus, designated Chinstrap penguin adenovirus 1 (CSPAdV-1), showed nucleotide (amino acid) sequence identity of 71.8% (65.5%) with South Polar skua 1 (SPSAdV-1), 71% (70%) with raptor adenovirus 1 (RAdV-1), 71.4% (67.6%) with turkey adenovirus 3 (TAdV-3) and 61% (61.6%) with frog adenovirus 1 (FrAdV-1). Based on the genetic and phylogenetic analyses, CSPAdV-1 was classified as a member of the genus, Siadenovirus. Virus isolation attempts from kidney homogenates in the MDTC-RP19 (ATCC® CRL-8135™) cell line were unsuccessful. In conclusion, this study provides the first evidence of new adenovirus species in Antarctic penguins.

The genome of Muju virus (MUJV), identified originally in the royal vole (Myodes regulus) in Korea, was fully sequenced to ascertain its genetic and phylogenetic relationship with Puumala virus (PUUV), harbored by the bank vole (My. glareolus), and a PUUV-like virus, named Hokkaido virus (HOKV), in the grey red-backed vole (My. rufocanus) in Japan. Whole genome sequence analysis of the 6544-nucleotide large (L), 3652-nucleotide medium (M) and 1831-nucleotide small (S) segments of MUJV, as well as the amino acid sequences of their gene products, indicated that MUJV strains from different capture sites might represent genetic variants of PUUV, the prototype arvicolid rodent-borne hantavirus in Europe. Distinct geographic-specific clustering of MUJV was found in different provinces in Korea, and phylogenetic analyses revealed that MUJV and HOKV share a common ancestry with PUUV. A better understanding of the taxonomic classification and pathogenic potential of MUJV must await its isolation in cell culture.

The recent discovery of genetically distinct hantaviruses in multiple species of shrews and moles prompted a further exploration of their host diversification by analyzing frozen, ethanol-fixed and RNAlater®-preserved archival tissues and fecal samples from 533 bats (representing seven families, 28 genera and 53 species in the order Chiroptera), captured in Asia, Africa and the Americas in 1981–2012, using RT-PCR. Hantavirus RNA was detected in Pomona roundleaf bats (Hipposideros pomona) (family Hipposideridae), captured in Vietnam in 1997 and 1999, and in banana pipistrelles (Neoromicia nanus) (family Vespertilionidae), captured in Côte d’Ivoire in 2011. Phylogenetic analysis, based on the full-length S- and partial M- and L-segment sequences using maximum likelihood and Bayesian methods, demonstrated that the newfound hantaviruses formed highly divergent lineages, comprising other recently recognized bat-borne hantaviruses in Sierra Leone and China. The detection of bat-associated hantaviruses opens a new era in hantavirology and provides insights into their evolutionary origins.

Small mammal surveillance was conducted (2008-2010, 2012) at Camp (Cp) Humphreys, a US Army installation and new expansion site, Republic of Korea (ROK), to identify hemorrhagic fever with renal syndrome health threats to US military/civilian populations during its ongoing expansion phase. Small mammals were collected using Sherman live capture traps and transported to Korea University where they were euthanized, tissues removed, and assayed to determine hantavirus IgG antibody-positive and hantavirus-positive rates by RT-PCR. A total of 2,364 small mammals were captured over 11,300 trap nights (capture rate = 20.92%). Apodemus agrarius was the most commonly collected (76.65%), with capture rates of 9.62% and 21.70% for Cp Humphreys and the expansion site, respectively. Overall, Hantaan virus (HTNV) IgG antibody-positive (Ab+) rate for A. agrarius was 2.15% (39/1,812). A total of 5.43% (10/184) Crocidura lasiura, 0.79% (2/254) Microtus fortis and 2.44% (1/41) Micromys minutus were serologically IgG Ab+ for hantaviruses. HTNV-specific RT-PCR demonstrated that 28.2% (11/39) HTNV Ab+ A. agrarius harbored the 328-nt sequence of the GC glycoprotein-encoding M segment of HTNV. Among them, the whole genome sequences of 3 HTNV strains were obtained by conventional RT-PCR and Rapid Amplification cDNA Ends PCR. Phylogenetic analyses of the HTNV strains from Cp Humphreys and the expansion site, Pyeongtaek, show a greater diversity of rodent-borne hantaviruses compared to HTNV previously identified in Gyeonggi province of the ROK. Thus, this study provides significant insights for raising HFRS threat awareness, analysis, and risk reduction strategies in southern Gyeonggi province.

Seasonal influenza is caused by two influenza A subtype (H1N1 and H3N2) and two influenza B lineage (Victoria and Yamagata) viruses. Of these antigenically distinct viruses, the H3N2 virus was consistently detected in substantial proportions in Korea during the 2010/11-2013/14 seasons when compared to the other viruses and appeared responsible for the influenza-like illness rate peak during the first half of the 2011/12 season. To further scrutinize possible causes for this, we investigated the evolutionary and serological relationships between the vaccine and Korean H3N2 strains during the 2011/12 season for the main antigenic determinants of influenza viruses, the hemagglutinin (HA) and neuraminidase (NA) genes. In the 2011/12 season, when the number of H3N2 cases peaked, the majority of the Korean strains did not belong to the HA clade of A/Perth/16/2009 vaccine, and no Korean strains were of this lineage in the NA segment. In a serological assay, post-vaccinated human sera exhibited much reduced hemagglutination inhibition antibody titers against the non-vaccine clade Korean H3N2 strains. Moreover, Korean strains harbored several amino acid differences in the HA antigenic sites and in the NA with respect to vaccine lineages during this season. Of these, the HA antigenic site C residues 45 and 261 and the NA residue 81 appeared to be the signatures of positive selection. In subsequent seasons, when H3N2 cases were lower, the HA and NA genes of vaccine and Korean strains were more phylogenetically related to each other. Combined, our results provide indirect support for using phylogenetic clustering patterns of the HA and possibly also the NA genes in the selection of vaccine viruses and the assessment of vaccine effectiveness.

BACKGROUND:Hantaan virus (HTNV), a negative sense tripartite RNA virus of the Family Bunyaviridae, is the most prevalent hantavirus in the Republic of Korea (ROK). It is the causative agent of Hemorrhagic Fever with Renal Syndrome (HFRS) in humans and maintained in the striped field mouse, Apodemus agrarius, the primary zoonotic host. Clinical HFRS cases have been reported commonly in HFRS-endemic areas of Gyeonggi province. Recently, the death of a member of the ROK military from Gangwon province due to HFRS prompted an investigation of the epidemiology and distribution of hantaviruses in Gangwon and Gyeonggi provinces that border the demilitarized zone separating North and South Korea. METHODOLOGY AND PRINCIPAL FINDINGS:To elucidate the geographic distribution and molecular diversity of HTNV, whole genome sequences of HTNV Large (L), Medium (M), and Small (S) segments were acquired from lung tissues of A. agrarius captured from 2003-2014. Consistent with the clinical incidence of HFRS established by the Korea Centers for Disease Control & Prevention (KCDC), the prevalence of HTNV in naturally infected mice in Gangwon province was lower than for Gyeonggi province. Whole genomic sequences of 34 HTNV strains were identified and a phylogenetic analysis showed geographic diversity of the virus in the limited areas. Reassortment analysis first suggested an occurrence of genetic exchange of HTNV genomes in nature, ROK. CONCLUSION/SIGNIFICANCE:This study is the first report to demonstrate the molecular prevalence of HTNV in Gangwon province. Whole genome sequencing of HTNV showed well-supported geographic lineages and the molecular diversity in the northern region of ROK due to a natural reassortment of HTNV genomes. These observations contribute to a better understanding of the genetic diversity and molecular evolution of hantaviruses. Also, the full-length of HTNV tripartite genomes will provide a database for phylogeographic analysis of spatial and temporal outbreaks of hantavirus infection.

Four US soldiers acquired hemorrhagic fever with renal syndrome while training near the Demilitarized Zone, South Korea, in 2005. Hantaan virus sequences were amplified by reverse transcription–PCR from patient serum samples and from lung tissues of striped field mice (Apodemus agrarius) captured at training sites. Epidemiologic investigations specified the ecology of possible sites of patient infection.

Antarctica is considered a relatively uncontaminated region with regard to the infectious diseases because of its extreme environment, and isolated geography. For the genetic characterization and molecular epidemiology of the newly found penguin adenovirus in Antarctica, entire genome sequencing and annual survey of penguin adenovirus were conducted. The entire genome sequences of penguin adenoviruses were completed for two Chinstrap penguins (Pygoscelis antarctica) and two Gentoo penguins (Pygoscelis papua). The whole genome lengths and G+C content of penguin adenoviruses were found to be 24,630-24,662 bp and 35.5-35.6%, respectively. Notably, the presence of putative sialidase gene was not identified in penguin adenoviruses by Rapid Amplification of cDNA Ends (RACE-PCR) as well as consensus specific PCR. The penguin adenoviruses were demonstrated to be a new species within the genus Siadenovirus, with a distance of 29.9-39.3% (amino acid, 32.1-47.9%) in DNA polymerase gene, and showed the closest relationship with turkey adenovirus 3 (TAdV-3) in phylogenetic analysis. During the 2008-2013 study period, the penguin adenoviruses were annually detected in 22 of 78 penguins (28.2%), and the molecular epidemiological study of the penguin adenovirus indicates a predominant infection in Chinstrap penguin population (12/30, 40%). Interestingly, the genome of penguin adenovirus could be detected in several internal samples, except the lymph node and brain. In conclusion, an analysis of the entire adenoviral genomes from Antarctic penguins was conducted, and the penguin adenoviruses, containing unique genetic character, were identified as a new species within the genus Siadenovirus. Moreover, it was annually detected in Antarctic penguins, suggesting its circulation within the penguin population.

Sequence analysis of the full-length medium segment and the partial small and large segments of a hantavirus, detected by reverse transcription–PCR in lung tissues of the Chinese mole shrew (Anourosorex squamipes) captured in Cao Bang Province, Vietnam, in December 2006, indicated that it is genetically distinct from rodentborne hantaviruses.

Rodent-borne disease surveillance was conducted at Nightmare Range (NM-R), near the demilitarized zone in northeast Gyeonggi Province, Republic of Korea, to identify hemorrhagic fever with renal syndrome (HFRS) risks for a mountainous high-elevation (500 m) military training site. Monthly surveys were conducted from January 2008-December 2009. A total of 1,720 small mammals were captured belonging to the Orders Rodentia [Families, Sciuridae (1 species) and Muridae (7 species)] and Soricomorpha [Family, Soricidae (1species)]. Apodemus agrarius, the primary reservoir for Hantaan virus (HTNV), accounted for 89.9% (1,546) of all small mammals captured, followed by Myodes regulus (4.0%), Crocidura lasiura (3.9%), Micromys minutus (1.4%), Mus musculus (0.3%), Microtus fortis (0.2%), Apodemus peninsulae (0.2%), Tamias sibiricus (0.1%), and Rattus norvegicus (

Antiviral drugs are being used for therapeutic purposes against influenza illness in humans. However, antiviral-resistant variants often nullify the effectiveness of antivirals. Combined medications, as seen in the treatment of cancers and other infectious diseases, have been suggested as an option for the control of antiviral-resistant influenza viruses. Here, we evaluated the therapeutic value of combination therapy against oseltamivir-resistant 2009 pandemic influenza H1N1 virus infection in DBA/2 mice. Mice were treated for five days with favipiravir and peramivir starting 4 hours after lethal challenge. Compared with either monotherapy, combination therapy saved more mice from viral lethality and resulted in increased antiviral efficacy in the lungs of infected mice. Furthermore, the synergism between the two antivirals, which was consistent with the survival outcomes of combination therapy, indicated that favipiravir could serve as a critical agent of combination therapy for the control of oseltamivir-resistant strains. Our results provide new insight into the feasibility of favipiravir in combination therapy against oseltamivir-resistant influenza virus infection.

Ticks comprising five species from the 'Hemaphysalis' spp. and 'Ixodes' spp. genera were screened through molecular techniques in the course of an investigation into the prevalence of tick-borne infectious agents in ticks in the Republic of Korea (ROK). A high rate of infection with 'Ehrlichia' and 'Anaplasma' sp. pathogens was demonstrated.

BACKGROUND:The discovery of genetically distinct hantaviruses in shrews (Order Soricomorpha, Family Soricidae) from widely separated geographic regions challenges the hypothesis that rodents (Order Rodentia, Family Muridae and Cricetidae) are the primordial reservoir hosts of hantaviruses and also predicts that other soricomorphs harbor hantaviruses. Recently, novel hantavirus genomes have been detected in moles of the Family Talpidae, including the Japanese shrew mole (Urotrichus talpoides) and American shrew mole (Neurotrichus gibbsii). We present new insights into the evolutionary history of hantaviruses gained from a highly divergent hantavirus, designated Nova virus (NVAV), identified in the European common mole (Talpa europaea) captured in Hungary. METHODOLOGY/PRINCIPAL FINDINGS:Pair-wise alignment and comparison of the full-length S- and L-genomic segments indicated moderately low sequence similarity of 54-65% and 46-63% at the nucleotide and amino acid levels, respectively, between NVAV and representative rodent- and soricid-borne hantaviruses. Despite the high degree of sequence divergence, the predicted secondary structure of the NVAV nucleocapsid protein exhibited the characteristic coiled-coil domains at the amino-terminal end, and the L-segment motifs, typically found in hantaviruses, were well conserved. Phylogenetic analyses, using maximum-likelihood and Bayesian methods, showed that NVAV formed a distinct clade that was evolutionarily distant from all other hantaviruses. CONCLUSIONS:Newly identified hantaviruses harbored by shrews and moles support long-standing virus-host relationships and suggest that ancestral soricomorphs, rather than rodents, may have been the early or original mammalian hosts.